Affinity modification of E 1-form of Na +, K + -ATPase revealed Asp-710 in the catalytic site

Abstract

An alkylating ATP analogue, γ-[4-( N-2-chlorethyl- N-methylamino)]benzylamide ATP (ClRATP), covalently binds to the catalytic α-subunit of Na +, K + -ATPase yielding a product resistant to hydrolysis by the enzyme and inhibiting the ATP-hydrolysing activity. The Na + -form of the membrane-bound Na +, K + -ATPase modified with ClRATP was hydrolysed by pepsin under conditions providing maximum stability of the modification product (4°C, pH 1.5). The modified peptide was isolated by HPLC and its amino acid sequence was found to involve residues 706–713 of the α-subunit polypeptide chain. This fragment located near the γ-phosphate of ATP is a component of the active site. It is highly homologous with corresponding regions of the catalytic subunits of all the known E 1-E 2 ATPases. In the Na + -(or E 1-)enzyme form Asp-710 is the target of modification. Evidently E 1- and E 2-enzymes have different targets in CIRATP modification, i.e. the polypeptide chain regions near the ATP γ-phosphate in the enzyme active site differ somewhat in their conformations.